A small-angle X-ray scattering study on pre-irradiated malate synthase. The influence of formate, superoxide dismutase, and catalase on the X-ray induced aggregation of the enzyme

Abstract

The sulfhydryl enzyme malate synthase from baker's yeast was X-irradiated with 6 kGy in air-saturated aqueous solution (enzyme concentration: congruent to 10 mg/ml; volume: 120 microliters), in the absence or presence of the specific scavengers formate, superoxide dismutase, and catalase. After X-irradiation, a small aliquot of the irradiated solutions was tested for enzymic activity while the main portion was investigated by means of small-angle X-ray scattering. Additionally, an unirradiated sample without additives was investigated as a reference. Experiments yielded the following results: X-irradiation in the absence of the mentioned scavengers caused considerable aggregation, fragmentation, and inactivation of the enzyme. The dose Dt37 for total (= repairable + non-repairable) inactivation resulted as 4.4 kGy. The mean radius of gyration was found to be about 13 nm. The mean degree of aggregation was obtained as 5.7, without correction for fragmentation. An estimation based on the thickness factor revealed that about 19% of material might be strongly fragmented. When this amount of fragments was accordingly taken into account, a value of 7.1 was obtained as an upper limit for the mean degree of aggregation. The observed retention of the thickness factor and the finding of two different cross-section factors are in full accord with the two-dimensional aggregation model established previously (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99-121 (1980)). The presence of catalytic amounts of superoxide dismutase and/or catalase, in the absence of formate, during X-irradiation reduced both aggregation and inactivation significantly. The presence of formate (10 or 100 mM) during X-irradiation led to a strong decrease of aggregation and inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)